PROJECT SUMMARY/ABSTRACT Long-term objectives and Specific Aims: This R21 proposal will extend recent advances in molecular microbiology to the rapid detection of pathogens in pneumonia. The long-term objective of this proposal is to accelerate the identification and quantification of pathogens in pneumonia using novel, real-time molecular technologies. In Aim 1, we will develop a protocol and pipeline that will rapidly identify respiratory pathogens via real-time metagenomics. In Aim 2, we will develop a protocol and determine reference ranges that will rapidly quantify respiratory pathogens using novel ultrasensitive PCR techniques. Research design and methods: We will develop these tools using bronchoalveolar lavage fluid that is prospectively collected from 200 patients undergoing bronchoscopy. The clinical status of these patients (healthy, chronically colonized, acutely infected) will be rigorously adjudicated. In Aim 1, we will perform real-time metagenomics using the MinION nanopore DNA sequencer. We will systematically optimize the speed and sensitivity of metagenomic identification of pathogens in bronchoalveolar lavage fluid. We will make publicly available: 1) a protocol for optimized real-time (4-hour) metagenomic identification of bronchoalveolar lavage fluid and 2) an informatic pipeline that rapidly discriminates host from microbial DNA without compromising protected patient information. We have recently demonstrated our ability to identify pathogens via this approach in nine hours. With optimization, we believe we can consistently achieve pathogen identification within four hours. In Aim 2, we will perform ultrasensitive PCR using two novel techniques, chiroplasmonic nanorod-PCR and droplet digital PCR. We will make publicly available: 1) a protocol for optimized rapid quantification of microbes in bronchoalveolar lavage fluid and 2) reference ranges of microbial burden in bronchoalveolar lavage fluid stratified by clinical status: uninfected, chronically colonized, and acutely infected. With optimization and the determination of clinical reference ranges, we believe we can accurately determine the microbial burden of respiratory specimens within two hours. Mission-relevance: Pneumonia remains a tremendous cause of morbidity, mortality, and healthcare expense. Our clinical identification of respiratory pathogens still relies on the culture-based techniques used by Pasteur in the 1880s. Delayed identification of pathogens in pneumonia results in increased morbidity and mortality, and indiscriminate use of broad empiric antibiotics impedes antimicrobial stewardship. The NIAID has identified a critical need for rapid, accurate identification of pathogens in pneumonia. This exploratory, novel study will extend recent discoveries in molecular microbiology to the rapid detection of pathogens in pneumonia. It represents a high-risk, high-reward study that may lead to a breakthrough in the diagnosis and management of pneumonia. It is not a clinical trial.